Protein identification has been revolutionised by the introduction of methods to identify proteins in databases using mass spectral data, either based on peptide masses from protein digests or fragmentation spectra from individual peptides. Interestingly, the main problem that is encountered in high sensitivity protein/peptide analysis and identification is usually not related to the sensitivity of the analysing device (usually a mass spectrometer, which can sequence peptides at the attomole level), but the generation and handling of peptides by digestion of the protein.
In the prior art, the target protein has usually been isolated by two-dimensional gel electrophoresis (2D-PAGE) or affinity chromatography and recovered in a fairly large volume of solution, such as about 20 μl for a spot from a two-dimensional electrophoresis gel. Thus, proteins have been recovered in a very diluted state. For example, if a microgram of material is available of a 50,000 MW protein, (20 pmol) the concentration is 1 μM. This is slightly below the Km of most proteases, and even though digestion can still take place, it will be a very slow process. Further, if only a nanogram is available, i.e. 20 femtomoles which is well within the sensitivity range of modern mass spectrometers using nanospray ionisation, digestion will not occur to any significant extent since then the protein solution is too dilute.
A further drawback of the prior art devices is that the container walls, which are usually of plastics or glass, absorb peptides and proteins very easily and large losses will occur within an hour or so of the protein/peptide solution coming into contact with the container. Furthermore, subsequent handling steps such as pipetting or being loaded into a chromatography system all involve contact with large surface areas and dramatically increasing sample losses.
For a general review of the above-mentioned methods, see e.g. Staudenmann, W., Dainese Hatt, P., Hoving, S., Lehmann, A., Kertesz, M., and James, P. (1998) Electrophoresis, 19, 901–908. “Sample handling for proteome analysis.”